Aim: The primary goal of this research was to create a therapeutic drug monitoring (TDM) approach for the oldest anticancer medication, 5-fluorouracil (5FU). Materials And Methods: We employed a Plasma matrix for analyte extraction through protein precipitation. Chromatographic separation of the analyte with the internal standard, 5-bromouracil (5BU), was carried out using a Phenomenex Luna C18 150*4.6mm 5um analytical column with an isocratic program. Gimeracil, which serves as a DPD enzyme inhibitor, was used to enhance the stability of 5FU in blood. The calibration curve for 5-fluorouracil was linear (r2 ≥ 0.99) from a range of 0.2μg/ml to 10μg/ml. The validation of the method was conducted following the bioanalytical method guidelines outlined by ICH and FDA in 2022. Conclusion: The method has been successfully developed and validated as a therapeutic drug monitoring (TDM) service to accurately determine the level of 5FU concentration in patients with gastric conditions, ensuring the specificity, sensitivity, and reliability of the method.